KR100808060B1 - Microorganism having fermentation activity of cervi parvum cornu and process for the preparation of fermentation product of cervi parvum cornu using the same - Google Patents

Abstract

A novel Bacillus licheniformis is provided to be able to prepare a fermented material of cervi parvum cornu with high yield, the fermented material having excellent anti-fatigue, the liver protective and diarrhea inhibitory effects. A Bacillus licheniformis with cervi parvum cornu fermenting activity is deposited as a deposition no. KCCM10885P. A method for preparing a fermented material of cervi parvum cornu comprises the steps of: (a) adding the Bacillus licheniformis to a culture solution including the cervi parvum cornu and fermenting it for 3-15 days; and (b) concentrating or drying the obtained fermented solution. Further, the drying step is performed by freezing drying.

Description

Microorganism having fermentation activity of cervi parvum cornu and process for the preparation of fermentation product of cervi parvum cornu using the same}

The present invention relates to a strain having a deer antler fermentation activity and a method for producing a deer antler fermentation using the same, more specifically, Bacillus rickenformis ( Bacillus) having a deer antler fermentation activity licheniformis ) and a method for producing a antler fermented product using the same.

Deer antler (Latin cervi parvum cornu) refers to the young, unosteophilized horn on the head of a plum, marok, or other dependent stag, belonging to the cervidae family. The rusted horn is called rusted horn, and the following year it is called a fall. Deer antler is divided into manok, plum, siberian, and NZ, and reindeer, depending on the type of mountain and deer.

In oriental medicine, deer antler has long been considered the best tonic tonic, and as described in Dong Bok-gam's literature, antler has tonic action, view blood action, gangjeong action, analgesic action, hematopoietic action, growth development promotion, heart failure treatment and hyperactivity. It is known to have various effects such as fatigue recovery, physical vitality enhancement, and renal function enhancement.

However, deer antler has side effects such as diarrhea, it is used in combination with herbal medicine with astringent action in the herbal, in which case the expected effect may be lost or reduced. In addition, antler, which is a useful resource of oriental medicine, is dipped in alcohol for use in Chinese medicine, and then used in small pieces. In this process, many active ingredients of antler are lost.

On the other hand, it is known that when the antler is fermented using a Bacillus strain or the like, a antler fermented product having an excellent immune enhancing effect can be obtained.

Korean Patent Registration No. 206,182 discloses Bacillus Py-92 strain (KCTC 0049 BP) having antler degradation activity isolated from animal intestinal contents or fresh stools (KCTC 1994; 25: 233-237, about Journal 12994; 38: 795-799). In addition, Republic of Korea Patent No. 329,365 discloses a antler solvent and a method of manufacturing the same by increasing the absorption rate by fermenting the antler (or rust) together with the system. In addition, Republic of Korea Patent Publication No. 10-2006-0128117 No. 10-2006-0128117 was cultured for 5 to 7 days by implanting Bacillus P-92 strain in a antler medium made by putting the antler pulverized in about 10 to 60 times the amount of antler weight distilled water It has been disclosed a pharmaceutical composition for immuno-enhancing, intestinal function and cancer prevention and treatment containing the fermented antler extract obtained by the post sterilization and filtration process as an active ingredient.

In addition, Korean Patent Registration No. 753,776 discloses a fermented food prepared by adding an antler extract and a method of manufacturing the same.

However, when the antler is fermented using a conventional method, for example, Bacillus Py-92 strain (KCTC 0049 BP) isolated from animal intestinal contents or fresh stools, or the selected salary, It is low, and the pharmacological activity of the antler fermentation is not satisfactory.

The present inventors carried out various studies to develop a method for producing a antler fermented product having excellent pharmacological activity, and a recovery rate of the antler fermented product. As a result, when the antler is fermented using a strain newly isolated from rice bran or rice bran, not only can the yield (i.e., recovery) be significantly increased compared to the conventional strain, but the obtained antler fermented product has excellent pharmacological activity. I found that.

Therefore, an object of the present invention is to provide a novel strain having antler fermentation activity.

In addition, an object of the present invention is to provide a method for producing a antler fermented product using the strain.

According to one aspect of the present invention, Bacillus licheniformis KCCM10885P having antler fermentation activity is provided.

According to another aspect of the invention, (a) Bacillus rickeniformis ( Bacillus ) in a culture solution containing antler licheniformis ) fermentation by adding KCCM10885P; And (b) it provides a method for producing a antler fermentation comprising the step of concentrating or drying the obtained fermentation broth.

In the production method of the present invention, the culture solution containing the antler can be preferably used by dispersing the antler in an aqueous medium ( Bacillus licheniformis) ( Bacillus licheniformis ) KCCM10885P is Bacillus rickeniformis ( Bacillus) licheniformis ) can be added in the form of rice bran or rice bran containing KCCM10885P. In addition, the rice husk or rice bran can be preferably used for 10 to 30 minutes treated at 80 ~ 120 ℃.

The fermentation is preferably performed for 3 to 15 days, preferably about 10 days, and the drying is preferably performed by lyophilization.

Bacillus licheniformis KCCM10885P, newly isolated according to the present invention, enables the production of a high yield of antler fermentation. In addition, the antler fermentation product obtained from the above strain has excellent anti-fatigue effect, hepatoprotective effect, diarrhea inhibitory effect and the like compared to the antler fermentation products using conventional strains.

The present invention includes Bacillus licheniformis KCCM10885P having antler fermentation activity.

The present inventors searched a variety of microorganisms having antler fermentation activity from rice bran and rice bran around the country. In other words, the rice husk and rice bran was treated for 30 minutes at 100 ℃ and suspended in TSB (tryptic soy broth), the supernatant was transplanted to TSA (tryptic soy agar) medium, and then incubated at 37 ℃ for 24 hours to grow colonies Deer Antler was added to search for strains with antler fermentation activity.

As a result of the search, about 20 strains having antler fermentation activity were selected. Among them, the highest fermentation yield (ie, recovery rate of antler fermentation) was selected, and strains which had no off-flavor were selected and named as KH-07 strains. It was.

The KH-07 strain was Gram positive bacilli by Gram staining. GDNA of KH-07 strain was isolated using Wizard genomic DNA purification kit (Promega, USA), primer [forward: 5'-TCA CCA AGG CRA CGA TGC G-3 '(SEQ ID NO: 2), reverse: 5' PCR [5 min at 94 ° C. using CGT ATT CAC CGC GGC ATG-3 ′ (SEQ ID NO: 3)]; Thereafter, 30 cycles of 30 seconds (denaturation at 94 ° C., 40 seconds (annealing) at 54 ° C., and 90 seconds at 72 ° C.] were obtained to obtain a PCR product of 1100 bp, which was obtained using a T-easy vector (Promega). JM 109 (Promega) was transformed after ligation. Plasmids were again isolated from the positive clones and sequenced using T7 and SP6 primers.

GenBank The sequence of 16S rDNA of KH-07 strain obtained from the homology search Blast system is shown in SEQ ID NO: 1. Sequencing showed very similar characteristics to Bacillus subtilis and Bacillus licheniformis , and when compared directly with the two strains, the latter showed more similar characteristics but not identical (see Table 1 below). ).

% Homology with KH-07 strains Search (High Homorology Analysis) Align (full method) direct order homology Bacillus subtilis subtilis ) - 98 - Bacillus licheniformis licheniformis ) ATCC 14580 99 99 91

According to the present invention, the bacteriological properties of the newly isolated KH-07 strain were Bacillus genus Py-92 strain (KCTC 0049 BP), Bacillus subtilis ( Kcillus subtilis). subtilis ) KCTC1325 and Bacillus rickeniformis ( Bacillus) licheniformis ) compared with KCTC1026 is shown in Table 2 below.

Bacillus py-92 Bacillus subtilis KCTC1325 Bacillus licheniformis KCTC1026 Bacillus licheniformis KH-07 Shape Rod Rod Rod Rod Gram Positive Positive Positive Positive Voges-Prokauer test + + + + Indole - - - - Methyl red + + + + Nitrate reduction - - - - Simmon's citrate + + + + Oxidase + + + + Urease + + + + Catalase + + + + Casein usability + + + + Gelatin Usability + + + + Starch Availability + + + + D-Glucose Availability + + + + L-Arabinose Availability + + + + D-Xylose Availability + + + + Amygdalin degradation - - - + Aerobic growth + + + + Anaerobic growth - - + +

KH-07 strains from the above bacteriological properties were determined to be known strains and novel strains, and in particular, as shown in the following examples, the antler fermented products exhibited excellent recoveries of antler fermentation products compared to conventional strains. Anti-fatigue effect, hepatoprotective effect, diarrhea inhibitory effect.

Based on the above results, the present inventors deposited the newly isolated KH-07 strain on the Korea Microbiological Preservation Theta (KCCM) on October 17, 2007, and received an accession number of KCCM 10885P.

The present invention includes a method for producing antler fermented product using KH-07 strain. That is, the present invention (a) Bacillus licheniformis ( Bacillus licheniformis ) KCCM10885P step of fermentation in the culture solution containing antler; And (b) comprises a method for producing a antler fermentation comprising the step of concentrating or drying the obtained fermentation broth.

As used herein, the term "deer antler" refers to a young horn that is not ossified in the head of plum, marok or other dependent stag, belonging to a cervidae, and includes all kinds of rust, fall, etc. unless otherwise indicated. do. The antler may include all those ground or dried by cutting in a conventional manner or dipped in alcohol.

In the production method of the present invention, the culture solution containing the antler may be preferably one obtained by dispersing the antler in an aqueous medium, for example, water. The amount of the aqueous medium such as water is not particularly limited, and for example, water of about 10 times the weight of antler may be used.

Bacillus rickenformis ( Bacillus) licheniformis ) KCCM10885P can be added as is or in starter form. When the cells are added as it is, the content of the cells can be added, for example, 100,000 to 1 million, but is not significantly limited.

In addition, Bacillus rickenformis ( Bacillus) licheniformis ) When KCCM10885P is added in the form of a starter, the strain may be added by inoculating chaff or rice bran. The starter can be obtained by adding about 1 × 10 ⁵ Bacillus licheniformis KCCM10885P to 10 g of conventional rice husk or rice bran, but the number of strains inoculated is not greatly limited. Preferably, when the starter-type chaff or rice bran is used after treatment at 80 to 120 ° C. for 10 to 30 minutes, the yield of the antler can be increased, and an unpleasant smell and taste can be reduced during fermentation.

The fermentation may be performed at room temperature (ie, about 25 ° C.) or about 37 ° C., and may be performed for 3 to 15 days, preferably about 10 days.

The production method of the present invention comprises the step of concentrating or drying the fermentation broth obtained as described above (step (b)). The concentration may be a conventional method such as concentrated under reduced pressure, the drying may be a method such as reduced pressure drying, freeze drying. Preferably the drying may be carried out by lyophilization.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the invention, but not to limit the scope of the invention.

Example  One. KH -07 Fermentation of Deer Antler Using Rice Bran and Rice Bran Inoculated with Strain

1 X 10 ⁴ KH-07 strains were inoculated into 1 g of rice husk or rice bran and treated as is (ie room temperature) or in an oven at 60 ° C., 80 ° C., 100 ° C., 120 ° C. for 30 minutes each. 10 times of water was added to 100 g of cut antler, and the inoculated rice hull or rice bran inoculated with the KH-07 strain prepared above was incubated at 37 ° C for 10 days. The culture solution was filtered to remove solids and lyophilized to prepare a antler fermented product.

The results of measuring the yield of the antler fermentation and the degree of unpleasant odor and taste according to the respective treatment conditions are shown in Table 3 below. Yield of antler antler was calculated by fermenting antler, filtering and lyophilization of raw antler.

Yield of Deer Antler Ferment (%) Unpleasant smell and taste Room temperature 60 ℃ 80 ℃ 100 ℃ 120 ℃ Room temperature 60 ℃ 80 ℃ 100 ℃ 120 ℃ chaff 19 20 28 31 29 +++ ++ + - - Rice bran 17 19 25 34 30 +++ + - - -

-none; + Weak; ++, normal; +++, strong

Example  2. KH -07 Fermentation of Deer Antler Using Rice Bran and Rice Bran Inoculated with Strain

Except for using the fragmented rust rust instead of the fragmented antler was carried out in the same manner as in Example 1 to prepare a antler fermented product, the results of measuring the yield and unpleasant smell and taste of the antler fermentation according to the respective treatment conditions Is shown in Table 4 below. Yield of antler antler was calculated by fermenting antler, filtering and lyophilization of raw antler.

Yield of Deer Antler Ferment (%) Unpleasant smell and taste Room temperature 60 ℃ 80 ℃ 100 ℃ 120 ℃ Room temperature 60 ℃ 80 ℃ 100 ℃ 120 ℃ chaff 7 12 15 22 21 +++ ++ - - - Rice bran 5 10 18 25 25 +++ + - - -

-none; + Weak; ++, normal; +++, strong

From the results of Examples 1 and 2 it can be seen that when fermented using chaff or rice bran as a starter, it is preferable to use for 10 to 30 minutes treatment at 80 ~ 120 ℃.

Example  3. Measurement of yield and pharmacological effect of antler extract

A test group (1st test group), which was extracted as it was on a water bath, and the extract was lyophilized, a test group (2nd test group) obtained by lyophilizing a fermentation broth obtained by fermentation using a Bacillus Py-92 strain, and a sediment Test group obtained by lyophilizing the fermentation broth obtained by carrying out the fermentation using a test group (third test group), and a test group obtained by lyophilizing the fermentation broth obtained by performing fermentation using a chaff inoculated with the KH-07 strain as a starter ( 4th test group), a test group obtained by lyophilizing a fermentation broth obtained by fermentation using a rice bran inoculated with KH-07 strain as a starter (5th test group), and the KH-07 strain was directly added to carry out fermentation. The yield and pharmacological effects were measured for the test group (sixth test group) obtained by freeze-drying the fermentation broth. Each test group is specifically as follows.

1st test group: Extract obtained by adding 1 liter of water to deer antler (100 g), extracting in water bath for 4 hours, and freeze drying (yield 4.1%)

Second test group: Fermented product obtained by adding 1 liter of water to deer antler (100 g), adding Bacillus Py-92 strain (1 X 10 ⁴ strains), fermenting for 10 days, sterilizing, and filtering and filtering the filtrate. Yield 17%)

3rd test group: Fermented product obtained by adding 1 liter of water to deer antler (100 g), adding sediment (100 g), fermenting at 42 ° C. for 10 days, sterilizing, filtration and lyophilization (yield 15%).

4th test group: 1 liter of water was added to deer antler (100 g), 1 g of rice hulls (treated for 30 minutes at 100 ° C) inoculated with 1 X 10 ⁴ strains of KH-07 strain and fermented at 37 ° C for 10 days. , Fermented product obtained by lyophilization of supernatant (yield 31%)

Test group 5: 1 liter of water was added to antler (100 g), and 1 g of rice bran (treated for 30 minutes at 100 ° C.) inoculated with 1 × 10 ⁴ KH-07 strains was fermented at 37 ° C. for 10 days. Fermented product obtained by lyophilization of supernatant (yield 35%)

6th test group: Fermented product obtained by adding 1 liter of water to deer antler (100 g) and adding 1 × 10 ⁴ strains of KH-07 strains for 10 days at 37 ° C. and freeze-drying the supernatant (yield 34%).

(1) yield measurement

Recovery rate (yield) of the antler extract for the fermented products of the first to sixth group is shown in Table 5 below.

Yield (%) 1st test group 4.1 2nd test group 17 3rd test group 15 4th test group 31 5th test group 35 6th test group 34

(2) Antifatigue  effect

A 25 x 40 x 17 cm transparent plastic container was filled with distilled water up to 14 cm and the swimming time was measured simultaneously in 10 swimming baths while maintaining the temperature of the thermostat at 18 ° C. In the swimming experiment, weights, weights of 8% were attached to the back of the neck, fixed to the back of the neck, physiological saline-controlled, and groups administered with each sample were allowed to swim at the same time 30 minutes after oral administration. And, the end time of swimming was calculated by measuring the swimming time until the swimming is enough to sink to the nose enough to be submerged under the surface of the water as a swimming time. The blood of the terminated mice was immediately taken and the amount of creatine in the blood was measured. Each group was made up of 10 rats and the samples were administered 20 mg or 50 mg per kg. The results are shown in Table 6 below.

Administration group Dose (mg / kg) Swimming time (minutes) Creatine in the blood Normal - - 0.58 ± 0.01 Control group (physiological saline) - 9 ± 0.4 0.69 ± 0.02 1st test group 20 12 ± 2.3 0.66 ± 0.01 50 14 ± 2.2 0.66 ± 0.01 2nd test group 20 11 ± 2.8 0.67 ± 0.01 50 12 ± 3.1 0.66 ± 0.02 3rd test group 20 12 ± 1.9 0.65 ± 0.01 50 14 ± 2.5 0.66 ± 0.01 4th test group 20 15 ± 1.9 0.63 ± 0.01 50 19 ± 3.2 0.62 ± 0.02 5th test group 20 16 ± 2.2 0.61 ± 0.02 50 20 ± 2.1 0.61 ± 0.02 6th test group 20 15 ± 1.5 0.63 ± 0.01 50 21 ± 2.5 0.62 ± 0.01

As can be seen from the results of Table 6, the antler fermentation prepared according to the present invention has an excellent anti-fatigue effect compared to the antler fermentation prepared using a conventional strain.

(3) hepatic damage protection effect

Five rats (Woongseong, 180-200 grams) were administered orally with 1 ml of 20% carbon tetrachloride (diluted with olive oil) per 100 grams. Administered. 24 hours after the administration of carbon tetrachloride, the blood was collected from the heart and the concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. The results are shown in Table 7 below.

Dosage (mg / kg) Blood concentration (Karmen unit) AST ALT Normal - 520 ± 23 212 ± 98 Carbon tetrachloride treated group (liver damage group) - 2455 ± 205 1352 ± 176 Carbon tetrachloride and first test group treatment 100 oral administration 1652 ± 238 1021 ± 134 Carbon tetrachloride and second test group treatment 100 oral administration 1543 ± 281 1082 ± 155 Carbon tetrachloride and third test group treatment 100 oral administration 1421 ± 197 972 ± 151 Carbon tetrachloride and fourth test group treatment 100 oral administration 1124 ± 222 721 ± 120 Carbon tetrachloride and fifth test group treatment 100 oral administration 1021 ± 212 688 ± 188 Carbon tetrachloride and sixth test group treatment 100 oral administration 1112 ± 218 635 ± 178 Carbon tetrachloride and silymarin treatment 100 oral administration 1422 ± 250 816 ± 211

From the results of Table 7 above, as a result of causing liver damage to rats with carbon tetrachloride, blood ALT and AST increased, but liver damage was improved in the antler and fermented antler treated groups, but fermented with rice hull, rice bran or KH-07 isolated therefrom. It can be seen that the deer antler fermented product has a strong soy protective effect.

(4) acute toxicity test

Acute toxicity testing was performed using 6-week-old specific pathogen-free (SPF) SD rats. Five animals of each group were orally administered fermented antler extract of the present invention at doses of 0.5, 1 and 2 g / kg. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, and hematological and hematological examinations were performed.

As a result, there were no clinical symptoms or dead animals in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemical test and autopsy findings. However, diarrhea was observed in the first test group (deer antler) and the second test group (internal fermentation antler). As a result, the antler fermentation prepared according to the present invention did not show a change in toxicity even in rats up to 2 g / kg, and the minimum lethal dose (LD ₅₀ ) is determined to be a safe substance of 2 g / kg or more.

(5) diarrhea Incidence  And moisture content measurement

10 mice (ICR male, 20-22g) were used in one group, and 0.5g / kg or 1g / kg of antler and antler fermented in 0.5 ml of saline solution were administered, respectively, and the incidence of diarrhea and water content in feces were measured. . Forage was used as a control drug. The results are shown in Table 8 below.

Dosage (g / kg) Water content (%) Diarrhea Untreated group (normal group) - 68 0/5 1st test group 0.5 76 2/5 One 78 4/5 2nd test group 0.5 75 2/5 One 78 5/5 3rd test group 0.5 71 0/5 One 72 0/5 4th test group 0.5 68 0/5 One 71 0/5 5th test group 0.5 69 0/5 One 71 0/5 6th test group 0.5 70 0/5 One 71 0/5 sulphate of soda 0.5 81 5/5 One 85 5/5

* Diarrhea that is difficult to pick up with tweezers when the water content in the feces is more than 75%

(6) Synthesis of Pharmacological Effects

The results of the above pharmacological effects test are summarized in Table 9 below.

Antifatigue effect Hepatoprotective effect Diarrhea inhibitory effect Chaff Fermentation +++ +++ +++ Rice bran fermentation +++ +++ +++ KH-07 strain fermentation +++ +++ +++ Fertility Payment + + - (diarrhea) Bacillus PY-92 Fermentation + + + velvet + + - (diarrhea)

-, no effect

+, Weak; ++, normal; +++, strong

Example  4. Notice With Bacillus  compare

Add 1 liter of water to Deer Antler (100 g) and strain KH-07, Bacillus subtilis subtilis ) KCTC1325 and Bacillus licheniformis KCTC1026 strains were added to 1 × 10 ⁴ strains, respectively, and fermented at 37 ° C. for 10 days, and the supernatant was lyophilized to obtain a antler fermented product. The anti-fatigue effect was measured with the obtained antler fermented product in the same manner as in (2) above. The results of measuring the yield and the anti-fatigue effect are shown in Table 10 below.

Yield (%) Antifatigue effect (50mg / kg dose) KH-07 strain 34 21 ± 3 Bacillus subtilis subtilis ) KCTC1325 17 15 ± 2 Bacillus rickenformis licheniformis ) KCTC1026 25 17 ± 2

From the above results, it can be seen that the newly isolated KH-07 strain according to the present invention shows excellent recovery rate of antler fermentation, and the obtained antler fermentation exhibits excellent pharmacological effect compared to the conventional strains.

Attach sequence list electronic file  

Claims (7)

Bacillus Lee Kenny having fermentation activity antler formate miss (Bacillus licheniformis ) KCCM10885P. (A) fermentation by adding Bacillus licheniformis KCCM10885P in a culture solution containing antler; And (b) concentrating or drying the obtained fermentation broth. The method according to claim 2, wherein the culture solution containing the antler is obtained by dispersing the antler in an aqueous medium. The method of claim 2, wherein the Bacillus rickenformis ( Bacillus) licheniformis ) KCCM10885P is Bacillus rickeniformis ( Bacillus) licheniformis ) Method of producing a antler fermented antler, characterized in that it is added in the form of rice bran or rice bran containing KCCM10885P. The method according to claim 4, wherein the rice hull or rice bran is treated for 10-30 minutes at 80 ~ 120 ℃. The method of claim 2, wherein the fermentation is carried out for 3 to 15 days. The method of claim 2, wherein the drying is performed by lyophilization.